Method and apparatus for spore disruption and/or detection

ABSTRACT

A method and apparatus for spore disruption and/or detection is provided. The method involves irradiating a sample with laser light, conveniently ultraviolet radiation, to disrupt any spores present and collecting any disrupted material for analysis. The disruption can involve breaking the spore open to release intrasporal DNA which is useful for fast screening and detection equipment. The disrupted material may be collected in a collection chamber which can be flushed with an extraction fluid to collect the disrupted material. The sample is preferably concentrated in a nanovial prior to being irradiated to give sample enrichment.

This invention relates to a method and apparatus for disrupting sporesto aid subsequent analysis, especially to a method and apparatus forfast release of intrasporal DNA.

There is a growing need to be able to detect and identify spore formingbacteria. For example in the food industry there is a desire for rapidanalysis of food stuffs to detect the presence of any bacterial spores,such as Bacillus cereus, before they can germinate and spoil produceand/or cause illness. Equally there is a need for rapiddetection/identification of spores such as Bacillus anthracis.

The current gold standard for detection of spores is germination viaheat activation and outgrowth. However this process takes up to 48 hoursand requires skilled personnel and therefore is unsuitable for rapididentification.

More rapid tests exploit antibodies associated to the surface of thespores (exosporium) for detection. Handheld immunochromatographic testsare available but the sensitivity of such test are low.

Matrix Assisted Laser Desorption and Ionisation (MALDI) is a standardtechnique for transferring large biomolecules into the vapour phase formass spectrometric analysis. MALDI has been used to detect specificbiomarkers associated with the outer layers of the spores.

In a MALDI analysis the analyte of interest is mixed with a suitablematrix material and a solvent on a substrate. The solvent is thenevaporated to leave the analyte co-crystallised with the matrixmaterial. A pulsed UV laser source is then directed to irradiate thesample. The matrix material absorbs the laser light and a rapidtemperature increase causes disintegration of the matrix ejecting aplume of sample. The ejection plume is input to a mass spectrometer toanalyse the ionised biomolecules and hence the irradiation step isperformed in a high vacuum.

The various biomarkers associated with the exosporium can then beidentified. However the method is not good at discriminating betweendifferent Bacillus species as the origin and identity of the biomarkersmay be unclear and different species may have similar biomarkers. Toincrease the number of released biomarkers corona plasma discharge maybe used or sonication pre-treatments could be used but discriminationbetween species is still relatively poor.

Sonication may also be used to modify the surface of spores so as to aidsubsequent detection in an immunoassay, for instance immunoassaysinvolve the binding of an analyte to a specific antibody contained onthe surface of a sensor. Detection sensitivity can be improved bymodification of the surface of the species to be detected so as toimprove subsequent binding to the antibodies on the biosensor.

Another method of screening for spores is to completely disrupt thespore so as to release intrasporal DNA for subsequent analysis viapolymerase chain reaction (PCR) assays. For instance ultrasonication hasrecently been proposed to completely disrupt spores in ‘Belgrader P.;Hansford D.; Kovacs G. T. A.; Venkateswaran, K; Mariella, R.;Milanovich, F.; Nasarabadi, s.; Okuzumi, m; Pourahmadi, F.; Northrup, M.A. Analytical Chemistry 1999, 71, 4232-4236’. However the samples canrequire pretreatments of up, to 90 minutes and so far the amount ofintracellular DNA released has been low so the technique would notcurrently be sensitive enough for most applications.

It is therefore an object of the present invention to provide a methodand apparatus for disrupting spores for subsequent detection whichmitigates at least some of the aforementioned disadvantages.

Thus according to the present invention there is provided a method ofdisrupting any spores in a sample comprising the steps of irradiating asample with laser light so as to disrupt any spores present in thesample; and collecting any disrupted material.

The method according to the present invention therefore uses laserenergy to disrupt any spores present in the sample and then collects thedisrupted material for subsequent analysis. Thus the method of thepresent invention allows very rapid disruption of any present spores forsubsequent analysis—for instance screening for certain bacteria.

The method may involve enclosing the sample in a collection chamber andirradiating the sample within the collection chamber. This can provide asealed environment for spore disruption. The method may also be arrangedto irradiate the sample so as to disrupt any spores present in thesample and eject material into the collection chamber. This can allowfor efficient collection of the disrupted material within the collectionchamber. The collection step may comprise the step, subsequent toirradiating the sample, of flushing the sample within an extractionfluid.

Thus the present invention provides a rapid and simple means ofdisrupting spores in a sample. The irradiation may be sufficient toeject material into a collection chamber where it can be easilycollected for subsequent analysis. However the irradiated sample willalso contain disrupted material and flushing the sample with anextraction fluid will collect such material. It is therefore possible touse laser light which is intense enough to disrupt the spores but whichis below the threshold for ejecting material into the vapour phase.Depending upon the material used this could have advantages as a lessintense irradiation step may cause less damage to any material releasedfrom the spore.

As the material ejected into the collection chamber is collected forsubsequent analysis the environment for irradiation does not need to bein vacuo. Indeed the chamber could be at atmospheric pressure and couldjust contain air. This removes the need for vacuum pumps etc. and meansthat samples can be easily collected in situ. Of course the collectionchamber could, if required, be filled at least partly with another fluidother than air. It may even be possible to fill the collection chamberwith a liquid to aid transfer of ejected material to an analysischamber.

As used herein the term disruption may mean modification of the surfaceof the spore in a similar manner as described above with reference tosonication. In some embodiments however it is preferable to completelydisrupt the spore so as to release the DNA contained within the spore.Intracellular DNA can be used to uniquely identify the particularbacterium by any known method such as PCR. Conveniently therefore theirradiation step is arranged to disrupt the spore so as to ejectmaterial from within the spore, such as DNA, into the collectionchamber.

Conveniently the sample is disposed within a matrix material. Disposingthe sample within a matrix material can aid in energy transfer from thelaser as the matrix material can be arranged to hold any spores in aspecified location and to absorb the incident radiation and undergo anexplosive type decomposition to disrupt the spores. Conveniently thematrix material is chosen so that it has maximum disruptive effect butdoes not destroy the released material of interest. Where it is desiredto release intrasporal DNA it has been found that matrix materials usedin standard MALDI techniques where DNA is the sample material areadvantageous, resulting in complete disruption of the spore but nodamage to the released DNA.

In some cases however it may not be necessary to use a matrix materialand the sample alone could be irradiated.

Conveniently the method includes the step of locating the sample on asubstrate. Conveniently this involves mixing the sample, and any matrixmaterial, with a solvent. The solvent mixture is then placed on thesubstrate and the solvent evaporated to leave the sample. Where a matrixmaterial is used the sample is left co-crystallised with the matrixmaterial.

Preferably the step of locating the sample on the substrate comprisesthe step of locating the sample in at least one micro-structured vial onthe substrate. Locating the sample in a micro-structured vial on thesubstrate tends to localise the sample in one place providing inherentenrichment of the sample which can increase the sensitivity of theprocess. Further as the sample is located in the micro-structured vial ahigh power laser can be focussed just on the micro-structured vial tocause spore disruption resulting in enhanced disruption. Convenientlythe sample is mixed with a solvent as discussed and applied to themicro-structured vial. Upon solvent evaporation the sample remainspinned to the micro-structured vial due to surface tension. The samplethen crystallises into the vial. Given that the solvent-sample dropletwill be larger than the micro-structured vial the method thereforeenriches the sample in the micro-structured vial. Repeating the processwith more drops provides yet further enrichment of sample.

The at least one micro-structured vial may have a volume of less than100 nL or less than 10 nl or less than 1 nl or less than 0.1 nL.

Preferably the method involves locating samples in a plurality ofmicro-structured vial on the substrate. The plurality ofmicro-structured vial may be arranged as an array. In use eachmicro-structured vial may be arranged to be co-located with a separatecollection chamber thereby allowing several samples of material to bedisrupted and collected separately, say by sequential irradiation by alaser. Alternatively a parallel illumination arrangement could be usedif desired. This allows a plurality of disrupted samples to becollected. In this way the same assay may be performed several times toverify results and reduce susceptibility to error. Additionally oralternatively more than one assay may be performed on the material so asto improve detection and identification.

Preferably the wavelength of illuminating radiation is substantiallymatched to the absorption maximum of the chosen matrix material, whenused. In other words the skilled person will be aware of the absorptionmaximum for the chosen matrix material, i.e. the wavelength at whichradiation is most strongly absorbed, and therefore the wavelength ofilluminating radiation is preferably chosen to be at or near to thismaximum. As mentioned above the matrix material may be selected for lowfragmentation of intrasporal DNA samples but could be also chosen toenable low fragmentation of spore coat proteins where these are theanalyte of interest for the chosen detection method. This would be ofinterest if one were to use uniquely identifiable proteins within theouter-exosporium layer of the spore for identification purposes as analternative to DNA analysis using PCR. Conveniently the wavelength ofillumination is within the ultraviolet range, for instance within therange 400-10 nm.

Preferably the sample is illuminated with pulses of laser radiation,having a duration in the range 1 ns to 100 ns. Conveniently relativelylow power laser illumination is used with power in the region of 75 kWpeak power. This gives pulse energies of 300 microjoules in energy andat a repetition rate of 35 Hz corresponds to an average power or 6 mW.

The method may also comprise an initial step of washing the sample priorto irradiating with laser light so as to remove low molecular weightacid soluble proteins associated with the outer spore layers, this couldrender the spore more susceptible to disruption by irradiation,especially irradiation with UV radiation. Spores can be coated withproteins which protect the spore from radiation such as UV radiation. Bypre-treating the sample to remove or reduce such protective proteins thepower and/or duration of laser illumination required to disrupt thespore can be reduced as compared to illuminating a sample without apre-treatment. This can allow shorter illumination times which may beuseful for ultra rapid detection systems. Conveniently the sample iswashed with an acidic aqeous based solvent to release and remove atleast some of the protective proteins. This could be achieved in asample pre-treatment step just prior to the laser irradiation.

The step of enclosing the sample within a collection chamberconveniently comprises the step of securing a collection plate to thesubstrate, the substrate and collection plate defining a cavity withinwhich the sample is located. By securing a collection plate to thesubstrate when the sample is illuminated the ejected material may beejected as a plume and will be deposited on the collection plate. Thecollected material could then be used in a detection step as will bedescribed later. Conveniently the collection plate has a microchannelformed therein and the microchannel at least partly forms the cavity ofthe collection chamber. Conveniently the microchannel is configured sothat it may be used as part of a microfluidic circuit—in this way thecollection plate may be removed from the substrate and used as part of amicrofluidic circuit to aid subsequent detection steps. In someembodiments the substrate and collection plate could together be used aspart of a microfluidic circuit.

The method may involve filling the collection chamber at least partlywith an extraction fluid, which may be water for instance, in order tobetter collect the ejected material. With the collection chamber atleast partly filled with an extraction fluid the ejected material is notonly more efficiently collected but the extraction fluid can be flushedthrough without needing to remove the original sample which reduces thepossibility of contamination. When a solid matrix material is used itmay be necessary to keep an air gap between the extraction fluid and thesample/matrix so as to prevent the sample dissolving. However liquidmatrix materials may be used in which case a liquid-liquid interfaceneeds to be maintained. Parts of the collection chamber may have surfacetreatments to keep the extraction fluid from mixing with the sample.

As mentioned the disrupted sample material can then be subjected to anyof a number of assays to detect and/or identify certain bacteria inspore form. Therefore in a second aspect of the present invention thereis provided a method for performing a test for the presence of certainspore forming bacteria in a sample comprising the steps of treating thesample so as to disrupt any spores present by applying the method of thefirst aspect of the invention and performing a test on the collectedejected material to determine the presence or otherwise of a knownbacteria.

Advantageously the collected material may be intrasporal DNA in whichcase the step of testing the collected material may involve performing aPCR based assay. As mentioned PCR assays on intrasporal DNA can uniquelyidentify the bacteria or detect the presence of particular bacteria. PCRanalysis is a well known and relatively quick analysis technique. Thedisruption method of the present invention is a very quick and efficientway of obtaining undamaged intrasporal DNA with relatively simpleequipment. Therefore the method according to the second aspect of theinvention offers an extremely quick and simple analysis equipment whichcan offer in situ analysis of material to determine the presence orotherwise of particular bacterial agents.

Where the ejected sample material is collected in a collection platehaving a microfluidic channel the detection method may involve the firststep of disrupting the sample according to the first aspect of theinvention, removing the collection plate from the substrate andintroducing it to a microfluidic circuit and performing the requiredassay in the microfluidic circuit.

As mentioned the method according to the first two aspects of theinvention provides for very fast spore disruption. The irradiation stepmay last for one second or less.

In a third aspect of the invention there is provided an apparatus fordisrupting spores located on a substrate comprising a collection platereleasably securable to the substrate to define a collection chamber anda laser apparatus arranged to illuminate a sample within the collectionchamber so as to disrupt any spores present in the. The apparatusaccording to the third aspect of the invention has all the advantagesdescribed above with reference to the first two aspects of theinvention. It provides a rapid and simple method of disrupting spores toaid subsequent detection or to release intrasporal material for use insubsequent assays.

The laser may be arranged to disrupt any spores present and ejectmaterial in the collection chamber. Conveniently the laser apparatuscomprises an ultraviolet laser. The laser preferably has an emissionwavelength matched to the excitation region of the absorbing matrixmaterial, ideally at the maximum absorption wavelength. Preferably thelaser is a pulsed laser.

Preferably the substrate has at least one micro-structured vial locatedtherein to hold sample material. As mentioned above use of amicro-structured vial can usefully contain sample material forirradiation and can also serve to enrich the sample giving enhancedsensitivity.

Preferably the collection chamber comprises a microchannel formed in thecollection plate and the collection plate can be used as part of amicrofluidic circuit.

The present invention could be usefully employed as a kit for detectionof certain bacterial agents. Therefore in a fourth aspect of the presentinvention there is provided a kit for screening for the presence orotherwise of spore forming bacteria comprising a substrate having atleast one micro-structured vial for holding a sample and a collectionplate relesably securable to the substrate, the substrate and collectionplate defining a collection chamber adapted, in use, to collect materialejected from a sample disposed in the micro-structured vial uponillumination with a laser. In use sample material is mixed with asolvent and deposited on the substrate over the micro-structured vial.The solvent then evaporates and surface tension keeps the samplematerial at the micro-structured vial. Generally a matrix material isalso mixed with the solvent and the sample and after the solvent hasevaporated the matrix material and sample are co-crystallised in themicro-structured vial. The kit therefore preferably comprises a solventand possibly a matrix material.

The kit may also comprise means for illuminating a sample disposed inthe collection chamber with laser light so as to disrupt any spores inthe sample and eject material into the collection chamber.

Preferably the kit also comprises a test means for performing an assayon the collected material. This test means preferably comprises amicrofluidic circuit which the collection plate can be releasablysecured to, either together with the substrate or having been detachedtherefrom. The test means may perform a PCR based assay on releasedintrasporal DNA. The kit according to the present invention thereforeprovides a simple and easy to use screening kit which can be used insitu and yield rapid results for detection or otherwise of known agents.

Part of the reason that the present invention is so useful is thebenefit gained through enriching the sample in a micro-structured vialprior to irradiation. Therefore according to a fifth aspect of theinvention there is provided a method of enriching a sample of materialcomprising the steps of dissolving the sample in a solvent, placing adroplet of solution on a micro-structured vial in a substrate andevaporating the solvent so as to crystallise the sample in themicro-structured vial. It should be noted that the terms dissolving andsolution should be read broadly. Depending on the analyte of interestthe sample may not actually be dissolve in the solution but may besuspended in a suspension. For example spore forming bacteria will notbe dissolved as such but will be held in suspension. However someanalytes may actually dissolve. Therefore throughout this specificationthe terms dissolve and suspend and solution and suspension should beread interchangeably depending upon the analyte of interest. Also itshould be noted that the enrichment process described with regard to thefifth aspect of the invention is applicable to a wide range of possibleanalytes and is not limited to spore forming bacteria. A similarenrichment effect would be achieved with a range of low abundancematerials where the surface chemistry of the target plate andmicro-structured vial are such so as to draw the sample into the vial.For instance when the substrate is PDMS the method would work well withanalytes that are hydrophobic in nature, for instance peptides/proteins.

Preferably the method involves adding a matrix material to the solutionso that the sample co-crystallises with the matrix material in themicro-structured vial. This allows a large droplet of sample material tobe crystallised in the micro-structured vial resulting in an enrichedsample as compared to that had a flat substrate been used. The enrichedsample may then be irradiated in one go by a high power laser with anarrow beam cross section resulting in efficient energy transfer andmaximising the amount of disrupted spore material that may be collected.

The method of enrichment may also involve the step of repeatedlyapplying a drop of solvent/material mix onto the micro-structured vialso as to further enrich the sample in the micro-structured vial.Preferably the sample is enriched by a factor of 10 or greater, 100 orgreater, 1000 or greater or 10,000 or greater.

The invention will now be described by way of example only with respectto the following drawings of which;

FIG. 1 shows a schematic of the steps of the method of the presentinvention,

FIG. 2 shows two alternative layouts for the collection chamber, and

FIG. 3 shows PCR results for a) a control sample of spores and b) asample of spores after being irradiated according to the presentinvention for 1 second.

Referring to FIG. 1 schematically is shown a laser based sporeenrichment and disruption process with integrated extraction of desorbedintrasporal content in a microchip format at atmospheric pressure.Amplification and identification of any released DNA may then beperformed in a second step by PCR. A sample containing, or thought tocontain, spores is first mixed with a laser light absorbing matrix and asolvent and a drop 2 of the mixture is applied onto a micro-structuredvial such as nanovial 4 in an elastomer plate 6—see FIG. 1 a. Uponsolvent evaporation the drop remains pinned to the nanovial 4 due tosurface effects and eventually crystallises into the nanovial. Thisallows for drop volumes much larger than the nanovial volume to beapplied, resulting in sample enrichment in the vial. A second elastomerplate which comprises a collection plate 8 with a microstructuredchannel 10 is then attached to the sample zone plate 6—FIG. 1 b.Referring now to FIG. 1 c, upon laser illumination 12 of the sample zonethrough the collection plate 8, the explosive disintegration of thematrix breaks open the protective spore layers. Simultaneously, releasedintrasporal content is ejected in a plume and deposited onto the innerwalls of the microchannel 10 in the collection plate 8. Since attachmentof the two plates is adhesion based, the top plate 8 can readily beremoved after illumination. Any released intrasporal DNA is thenrecovered by flushing the channel in the top plate 8 with water and thecollected volume is subjected to PCR—FIG. 1 d.

The process described above allows ultrafast spore disruption in thesecond to sub-second range. Furthermore, the sample enrichment andamplification in the PCR step yields high sensitivity. It should benoted however that other assays than PCR based assays could equally beapplied and that the disruption process may be designed to modify thesurface of the spores for subsequent analysis rather than break open theouter spore layers.

The first step, as mentioned, is to mix the sample with a suitablematrix material and a solvent. The choice of matrix material will dependupon the desired result but matrix materials known for Matrix AssistedLaser Desorption and Ionisation (MALDI) may be appropriate. Where it iswished to break open the spore to release intrasporal DNA a matrix knownfor MALDI DNA analysis may be used. For DNA analysis the most commonlyused matrix materials are 2,4,6-trihydroxyacetophenone (THAP),6-aza-2-thiothymine (ATT) and 3-hydroxypicolinic acid (3-HPA). It shouldbe noted however that when used in a MALDI apparatus it is necessarythat the resultant material be ionised. This is not necessary in thepresent invention and hence the choice of matrix material is lessconstrained. In principle any matrix which is soluble in an aqueousmedium and absorbs highly at the wavelength of operation will besufficient. 3-HPA was used in tests as a suitable candidate based on itssolubility in water and comparatively low fragmentation effects ontarget DNA. The laser source used was a nitrogen laser with a 4 nspulsed output at 337 nm. The emission wavelength of 337 nm is stronglyabsorbed by 3-HPA. The skilled person will appreciate however thatmaterials which could equally be used for a matrix which might havehigher absorption maxima and/or lower fragmentation effects on DNAsamples or may be designed to release other analytes and absorb atdifferent wavelengths suitable for excitation using other illuminationsources.

The energy density of the illuminating radiation will vary depending onthe optical set-up used for illumination. The skilled person will beaware of various optical arrangements that could be used. For instanceoptic fibre coupling or use of appropriate lenses (open beamconfiguration). A range of output energies of between 0.4 microJoule(equivalent to 0.04 mJ/cm2, which is below the threshold energy densityof ˜10 mJ/cm2 required for desorption and plume ejection—obtained usingthe optic fibre set-up) and 68 microjoules (energy density of 170mJ/cm2, obtained using the open beam configuration) were achieved.

It should be noted that prior art MALDI schemes for analysis of sporeshave not resulted in complete spore disruption and have concentrated onreleasing ionised biomarkers from the exosporium. As such the matrixmaterials mentioned above have not previously been regarded as suitablefor use with spores in a MALDI analysis.

It is also noted that spores contain dipicolinic acid in the outerlayers and as such a matrix material might not be required in allembodiments and absorption of laser light by the spores themselves maybe sufficient to cause disruption.

Solvent choice is governed by the need to maintain analyte suspensionand promote partitioning of the analyte into the matrix crystals duringdrying of the analyte/matrix mix. For DNA analysis the solvent of choiceis water (both DNA and the above mentioned matrices are water soluble).To reduce the drying time and to get a more uniform and dense crystaldeposit, more volatile organic solvents such as acetone or acetonitrilecan be added. However, the reduced surface tension of the resultingsolvent systems can adversely affect the confinement of the evaporatingdrop into the microstructured sample vial 4 (high surface tension keepsthe evaporating drop pinned to the vial, “corralling effect”). Samplespot confinement is important to get a well defined target for the laserbeam 12 and as will be described later is important in a two phase flowsystem for extraction of potentially released spore DNA.

Various experiments were conducted on solvent mixtures andconcentrations to determine the optimum conditions for application andevaporation of 0.1 μL of sample/matrix mix into the microstructuredsample vials. Reproducible, near-perfect confinement and dense, uniformdeposits were obtained for 10 mg/mL 3-HPA in 40% acetonitrile/60%aqueous. Further sample enrichment was attempted through multipleapplication/evaporation of droplets and through increasing the appliedvolume. While the former approach is hampered by deposits beingredissolved the latter approach looks promising for microlitre volumes.However, the matrix concentration has to be adjusted to the appliedvolume. Also there appears to be a limit to the sample concentrationthat can be analysed in this way. For proper crystal formation thematrix always has to be in around 1000× molar excess with respect to theanalyte.

The sample plate and collection plate may be fabricated frompolydimethylsiloxane (PDMS) by moulding from a photoresist master.However other methods may be more appropriate and the skilled personwould know of a range of methods that could be applied to fabricate theplates.

The microfluidic layout of the collection plate may vary depending uponthe required use as will the dimensions. By way of example only the mainchannel may be in the region of 5 mm long. The width of the main channelmay vary between 600-1000 μm, but might be more if an array of nanovialsis used. The depth may be approximately 10 μm to minimise the extractionvolume (main channel: 30-50 nL). The sample spot size, and hence thesize of the nanovial, is limited by the minimum diameter of the laserbeam. Using a commercially available nitrogen laser with a coupler andsuitable fibre optics and post fibre focussing optics a beam diameter ofthe order of 200 μm may be achieved with a power density ofapproximately 8 mJ cm⁻² (with a coupling loss of approximately 90%).Therefore sample vials with dimensions ranging from 200-500 μm weredesigned (volume: 0.4-1 nL). Sample vial arrays should allow multipleshots without need for repositioning of the microfluidic chip.

The exact design of chip will depend upon the means by which the ejectedmaterial is to be collected. It is possible to use a solid or liquidmatrix material with air in the rest of the collection chamber andcollect material on the inside walls of the chamber. In such anembodiment the sample zone and extraction liquid are arranged in avertical arrangement, as shown in FIG. 1, to exploit best directionalityof the plume (normal to the surface). After irradiation of the samplematerial will have been deposited on the walls of the channel 10 inplate 8. Collection plate 8 may then be detached from sample plate 6 andhave another flat plate attached as a seal (not shown). Water may thenbe fed into the channel through inlet 14 and out through outlet 16 toflush the deposited material out where it may be used in a subsequentanalysis.

As will be explained later however there are a variety of geometries forthe collection chamber and it may not be necessary to separate thesubstrate from the collection chamber to flush through with extractionfluid. Indeed it may be wished to collect the material from the nanovial4 in the extraction fluid as this will also contain disrupted material.In some embodiments the laser illumination is sufficient to disrupt thespores but is not sufficient to eject any material out of the nanovial,i.e. no material is volatilized. In this case all the disrupted materialwill be left in the nanovial following illumination. Illuminating belowthe threshold required to eject material into the vapour phase can beadvantageous where the material of interest, for instance intracellularDNA, is relatively fragile and could be damaged by intense irradiation.Prior art MALDI techniques all work on material ionised in the vapourphase and so illumination has to be above the threshold to ejectmaterial into the vapour phase.

In some embodiments then there is no need for a collection plate and thesample and any matrix material may be illuminated directly withdisrupted material being left in the nanovial for collection.

The method may also involve a pre-treatment step of washing the samplethought to contain spores to remove or reduce any radiation protectiveproteins, so called low molecular weight acid soluble proteins from thespore outer layers. As will be understood by one skilled in the art thespore layer may contain various proteins which act to protect the sporefrom radiation damage or disruption, such as UV radiation. Whenirradiated these proteins in the layer/s may serve to protect the spore.By pre-washing the sample with a suitable treatment, such as an acidicaqueous based solvent, the amount of protective proteins on the sporelayer/s can be reduced. This can reduce the power and/or duration ofradiation exposure needed to disrupt the spore. When the method is usedin a ultra rapid detection system for identifying dangerous pathogenicorganisms speed of detection is key and so a reduced illumination timemay be beneficial. The skilled person would be aware of possible lowmolecular weight protective proteins for the spores of interest andsuitable treatments to pre-wash the sample with.

FIG. 2 shows a range of possible geometries. FIG. 2 a shows thesituation where spores alone are irradiated in a nanovial with thedisrupted material being left in the nanovial for collection. The samearrangement would equally apply were the spore mixed with a matrixmaterial which may be solid or liquid.

Where a liquid matrix material is used, or the spores are disposed inwater, and it is desired to collect ejected material in a collectionchamber an arrangement similar to that shown with respect to FIG. 1 maybe used.

In some embodiments though it may be better to have the materialcaptured in liquid in the collection chamber. Where a solid matrixmaterial is used however it is necessary to keep an air interfacebetween the crystallised sample/material mix and any extraction liquidto prevent the sample/matrix from re-suspending. FIG. 2 b shows a layoutthat may be used when the matrix used is a solid. In this case the PDMSsample plate 6 would comprise a channel 20 with lower level samplenanovials 4. This sample plate 6 would be placed below a PDMS collectionplate 8 comprising a second channel 22. The PDMS material in the secondplate 8 is plasma-treated to improve its hydrophilicity (or anothertreatment such as silanisation could be employed). Introducing theaqueous liquid phase extraction fluid 30 into the assembled microchipwould then result in selective filling of the channel in the collectionplate due to the higher hydrophilicity. This should leave the channel 20on top of the sample vial filled with air 32, resulting in an air-liquidinterface. Illumination of the sample zone 4 through the sample platecould then be used to generate a plume directed towards the liquidextraction phase. This should greatly enhance the extraction yield.Alternatively using a reflection geometry the sample could beilluminated through the collection plate 8.

In another embodiment, shown in FIG. 2 c, a liquid phase UV matrix 34could be employed in a liquid/liquid configuration. Again illuminationwould be in a transmission geometry through the UV matrix. Such anarrangement could be implemented in a parallel or layered flowconfiguration (illumination from side or top, respectively).

Test Results

Using the apparatus shown in FIG. 1 experiments were performed to testfor spore disruption. 0.1 μL of 10⁸ cfu/mL Bacillus globigii spores wasmixed 1:1 with 10 mg/mL 3-HPA matrix in 40 v/v-% ACN/60% water and wasapplied onto a 200×200 μm nanovial with a 20 μm depth molded inPolydimethylsiloxane (PDMS). Upon solvent evaporation this resulted in a100-fold enrichment with ˜10,000 spores in the vial. In a first testexperiment the PDMS flowcell was not attached and the zone was directlyilluminated for 1 second at 30 Hz with a 6 mW 337 nm nitrogen laser viafibre optics (output ˜50 μJ/cm²). Desorption was not observed and thesample zone was redissolved in 1 μL sterile water and subjected to PCR.It should be noted that the spores were pre-treated with chloros (sodiumhypochlorite) prior to illumination to remove any extrasporal DNA.

First a negative control group of untreated spores was subjected to thePCR treatment. The results are shown in FIG. 3 a. Then a sample ofspores that had been illuminated for 1 second was analysed and theresults are shown in FIG. 3 b. The PCR product (78 bp) is highlighted40.

The results clearly show that release of intrasporal DNA occurred.Electron micrographs of the illuminated zone confirmed spore damage.

In order to also induce desorption the optical set-up was changed to anopen beam configuration with focusing optics yielding an output of ˜170mJ/cm². Experiments revealed complete desorption of the sample zonewithin seconds under atmospheric pressure to enable recovery ofintrasporal content with the geometries described above.

1. A method of disrupting any spores in a sample comprising the stepsof: irradiating a sample with laser light so as to disrupt any sporespresent in the sample and; collecting any disrupted material.
 2. Amethod as claimed in claim 1 wherein the method includes the step ofenclosing the sample within a collection chamber and the sample isirradiated within the collection chamber.
 3. A method as claimed inclaim 1 wherein the step of irradiating the sample involve irradiatingthe sample sufficiently so as to disrupt any spores present in thesample and eject material into the collection chamber.
 4. A method asclaimed in claim 3 wherein any ejected material is collected within thecollection chamber.
 5. A method as claimed in claim 1 wherein the stepof collecting any disrupted material involves the step, subsequent toirradiating the sample, of flushing the sample with an extraction fluid.6. A method as claimed in claim 1 wherein the step of irradiating thesample is performed at substantially atmospheric pressure.
 7. A methodas claimed in claim 1 wherein the step of irradiating the sampleinvolves irradiating the sample with laser light so as to sufficientlydisrupt the spore so as to release material from within the spore.
 8. Amethod as claimed in claim 1 wherein the method includes the step ofdisposing the sample within a matrix material prior to irradiation.
 9. Amethod as claimed in claim 1 wherein the method comprises the initialstep of locating the sample on a substrate.
 10. A method as claimed inclaim 9 wherein the step of locating the sample on a substrate comprisesdisposing the sample in a solvent, placing the solvent-sample mixture onthe substrate and evaporating the solvent.
 11. A method as claimed inclaim 9 wherein the step of locating the sample on the substratecomprises the step of locating the sample in at least onemicro-structured vial on the substrate.
 12. A method as claimed in claim11 wherein the step of locating the sample in at least onemicro-structured vial enriches the sample in the micro-structured vial.13. (canceled)
 14. A method as claimed in claim 11 wherein the substratecomprises a plurality of micro-structured vials and the method involvesthe step of locating a sample in each micro-structured vial.
 15. Amethod as claimed in claim 14 wherein the method involves enclosing eachmicro-structured vial in a separate collection chamber and separatelyirradiating each sample in a micro-structured vial.
 16. A method asclaimed in claim 2 wherein the method includes an initial step oflocating the sample on a substrate and wherein the step of enclosing thesample within a collection chamber comprises the step of securing acollection plate to the substrate.
 17. A method as claimed in claim 16wherein the collection plate has a microchannel formed therein, themicrochannel forming at least part of the collection chamber.
 18. Amethod as claimed in claim 17 wherein the microchannel is configured sothat it can be used as part of a microfluidic circuit.
 19. A method asclaimed in claim 2 wherein the collection chamber is at least partlyfilled with an extraction fluid to collect any disrupted material.
 20. Amethod according to claim 1 further comprising the step of performing atest on the collected material to determine the presence or otherwise ofbacteria.
 21. A method according to claim 20 wherein the collectedmaterial is intrasporal DNA and the test to determine the presence orotherwise of known bacteria comprises a PCR based assay.
 22. A methodaccording to claim 20 wherein the method includes an initial step oflocating the sample on a substrate and wherein the step of enclosing thesample within a collection chamber comprises the step of securing acollection plate to the substrate and wherein the step of performing atest on the collected material involves removing the collection platefrom the substrate, introducing it into a microfluidic circuit andperforming the test in a microfluidic circuit.
 23. A method as claimedin claim 1 wherein the step of irradiating the sample is performed for asecond or less.
 24. A method as claimed in claim 1 wherein theirradiating radiation has a wavelength in the ultraviolet range.
 25. Amethod as claimed in claim 1 wherein the irradiating radiation is pulsedradiation, each pulse having a duration of between ins and 100 ns. 26.An apparatus for disrupting spores located on a substrate comprising acollection plate releasably securable to the substrate to define acollection chamber and a laser apparatus arranged to illuminate a samplewithin the collection chamber so as to disrupt any spores present in thesample.
 27. An apparatus as claimed in claim 26 wherein the laser isarranged to disrupt any spores present and eject material into thecollection chamber.
 28. An apparatus as claimed in claim 26 wherein thelaser apparatus comprises an ultraviolet laser.
 29. An apparatus asclaimed in claim 26 wherein the substrate has at least onemicro-structured vial arranged therein for holding a sample.
 30. Anapparatus as claimed in claim 26 wherein the collection chamber is atleast partly formed from a microchannel formed in the collection plate.31. An apparatus as claimed in claim 30 wherein the collection plate canbe used as part of a microfluidic circuit.
 32. A kit for screening forthe presence or otherwise of spore forming bacteria comprising asubstrate having at least one micro-structured vial for holding a sampleand a collection plate relesably securable to the substrate, thesubstrate and collection plate defining a collection chamber adapted, inuse, to collect material released from a sample disposed in themicro-structured vial upon illumination with a laser.
 33. A kit asclaimed in claim 32 further comprising a store of solvent.
 34. A kit asclaimed in claim 32 further comprising a store of matrix material.
 35. Akit as claimed in claim 32 wherein the kit further comprises a means forirradiating a sample in the collection chamber with laser light so as todisrupt any spores in the sample.
 36. A kit as claimed in claim 32further comprising means for performing an assay on material collectedin the collection chamber.
 37. A kit as claimed in claim 36 wherein themeans for performing an assay comprises a microfluidic circuit withinwhich the collection plate can be releasably secured.
 38. A kit asclaimed in claim 36 wherein the means for performing an assay performs aPCR based assay on released intrasporal DNA. 39-42. (canceled)